Over the last 20 years there have been great advancements in protein synthesis, recombinant organism expression of complex molecules, particularly proteins, and hybridization of cells to produce monoclonal antibodies. These and other developments have lead to increasing need for apparatus and methods for efficiently separating desired products from co-products and various other contaminants.
Electrophoresis has been previously used for protein and peptide separation. However, electrophoresis has in general been performed at low flow rates not adapted to preparative scale production to provide significant quantities of the desired products. Many prior art electrophoretic separation systems utilize a confined chamber formed between two plates. An electric field is established between two opposing electrodes or in a desired two-dimensional array using additional electrodes. Such systems typically have good resolution of proteins but have limited flow rates. They are also typically limited to a single type of processing. They are further relatively complex and costly and troublesome to operate at preparative scale sizes. Preparative scale systems must be contrasted to laboratory scale systems used to discriminate proteins contained in samples for identification and extraction in very limited quantities.
One preparative scale electrophoresis apparatus is the ROTOFOR.RTM. by Bio-Rad Laboratories of Richmond, Calif. 94804. This apparatus performs preparative scale free solution isoelectric focusing of proteins using a rotating electrophoretic processing chamber. The rotating electrophoretic processing chamber has a horizontal axis of rotation which extends between the electrodes. The processing chamber is divided into twenty discrete compartments using membrane screens which are transversely placed along the horizontal axis. An ampholyte solution is used to create a pH gradient between the charged electrodes. The varying pH along the chamber allows proteins to selectively collect (focus) at the pH corresponding to their associated isoelectric points. Thus proteins having differing isoelectric points can in many cases be separated into one of the discrete compartments.
The ROTOFOR performs only in a batch mode of operation and only performs isoelectric focusing. The batch operation takes about 4 hours, thereby limiting the processing rates which can be achieved. This unit further must be stopped to extract the separated proteins and cannot be used in a continuous flow mode of operation. The separated fractions are removed using a harvesting apparatus having a series of tubes which tap the processing chamber at various fixed locations along the axis between the electrodes. This unit is also relatively costly to operate.
One apparatus of the current invention is advantageous in providing a construction having features which allow it to be used for several different types of electrokinetic separation. Electrophoresis can be performed in both batch and continuous flow modes of operation. Additionally, batch isoelectric focusing can be performed and extraction at various locations from the processing chamber can be accomplished. The system is relatively less complex than prior systems. It is also relatively low cost in both operation and initial construction of the system. It further provides improved heat transfer and resists problems due to electroosmosis while providing good separation of most proteins or other processed chemicals of varying electrophoretic mobilities.
Isoelectric electrophoresis processes use an ampholyte mixture to establish a pH gradient. Ampholytes and other buffer modifiers are expensive, thus increase processing costs. Another apparatus of this invention is advantageous in that electrophoretic processes can be carried out without use of expensive ampholyte buffers.